The long term goal of this application is to understand the molecular basis of presenilin (PS)-linked production of Ap40 and Ap42 in familial Alzheimer's disease (FAD). Very little is known about the structure of the PS/y-secretase complex due to the complexity and difficulty of crystallizing multipass transmembrane proteins. We hypothesize that PS/y-secretase exists in different conformations, leading to different alignments of APP with the catalytic site, resulting in the production of different AB species. Our observations, using a novel FRET based technique in intact cells as well as biochemical approaches;provide methods to examine PS1/y-secretase conformation in intact cells as well as to determine proximity of PS1 and y-secretase substrates, including APP and Notch. Our observations support a model with the following important, testable features: mature PS1 associates with other y-secretase complex members and adopts a conformation with N- and C-termini (NT and CT) close together;and the specific presentation of APP to the y-secretase active site changes under conditions favoring AB40 vs. AB42. We will test this model from several clinically important perspectives. FAD-linked PS1 mutations lead to elevations in Ap42/4o ratio, but the mechanism is unknown. Our preliminary data suggest that FAD PS1 mutations bring PS1 NT and CT even closer together, and that this represents a pathogenic change in conformation associated with an alteration in the alignment of the active site of PS1/y-secretase with APP, favoring cleavage at Ap42. We will test several models to define the precise molecular mechanism that leads to this observed change in conformation (Aiml). Some nonsteroidal anti-inflammatory drugs have been shown to selectively lower AB42, a phenotype opposite to FAD mutations. We will test the hypothesis that pharmacological agents that selectively alter the AB42/40 ratio can do so by allosterically modulating the conformation of PS1/y-secretase and PS1/APP interactions in the opposite way to FAD mutations. We will explore whether Ap42 lowering allosteric modulators of the y-secretase can "fix" the pathogenic FAD mutant PS1 conformation (Aim2). Finally, we propose to test the hypothesis that physiologic stimuli that activate y-secretase and/or its substrate selectively change PS1 /y-secretase interactions with substrates in neurons. We will take advantage of the high spatial resolution of the new FRET technique, Fluorescence Lifetime Imaging Microscopy (FLIM), which is uniquely suited for monitoring relative conformational changes of proteins and complexes in intact and/or live cells, to show in what subcellular compartments in intact cells these changes occur (Aim3), and ultimately aim to extend these studies to the mouse brain. The results obtained will provide important new insights into conformational changes in the PS1/y-secretase complexes and its interaction with substrates within a cellular context, and thus will help in the design of allosteric modulators of y-secretase function as a therapeutic approach. More generally, the assay of monitoring PS1 conformation and y-secretase -substrate proximity will help to help develop methodologies that may prove useful in understanding disease-related protein conformation changes.